DNA/RNA extractions from E5 project

Extractions from the three coral species from each of the four timepoints

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Extraction Date: September 23, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
41	January	Acropora	ACR-225	20200103	2
77	January	Pocillopora	POC-205	20200103	2
97	January	Porites	POR-367	20200106	3
303	March	Acropora	ACR-244	20200303	2
305	March	Pocillopora	POC-222	20200303	2
319	March	Porites	POR-236	20200303	2
605	Sept	Porites	POR-340	20200908	3
611	Sept	Pocillopora	POC-369	20200908	3
631	Sept	Pocillopora	POC-42	20200911	1
741	November	Porites	POR-72	20201101	1
757	November	Pocillopora	POC-52	20201101	1
765	November	Porites	POR-76	20201101	1

Extraction notes

• ACR and POC samples: pulled out 300ul of shield
• POR samples: pulled out 150ul of shield and added to 150ul of new shield
• Spun down samples for 3 minutes at 9000 rcf and then transfer the supernatant to new tube without disturbing the pellet
• 300ul of shield, 15ul of ProK, and 30ul of ProK digestion buffer, let sit for 2 minutes
• All spins were done for 1 minute or 2.30 minutes
• Did two washes with 700ul of wash buffer for both the DNA and RNA
• Then followed the protocol as described in protocol

Qubit

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA

Tube number	RFU	DNA 1 (ng/uL)	DNA 2 (ng/uL)	Average
Standard 1	196.76
Standard 2	21813.55
41		25.8	25.8	25.8
77		20.6	20.6	20.6
97		nd	nd	nd
303		49.6	49.2	49.4
305		84.8	84.8	84.8
319		3.50	3.50	3.50
605		3.46	3.40	3.43
611		3.92	3.84	3.88
631		96.8	97.0	96.9
741		2.64	2.60	2.62
757		50.2	50.2	50.2
765		5.34	5.34	5.34

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	358.25
Standard 2	6841.70
41		11.8	12.6	12.2
77		23.8	23.4	23.6
97		nd	nd	nd
303		12.4	12.8	12.6
305		33.8	34.4	34.1
319		26.6	27.6	27.1
605		24.6	24.4	24.5
611		46.8	46.0	46.4
631		62.4	62.6	62.5
741		27.6	28.4	28.0
757		33.6	33.6	33.6
765		30.4	31.0	30.7

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #97
• Results Link

Gel

• Modified from this protocol
• Added 0.75g of agarose and 50ml of 1x TAE to flask and microwaved for 45 seconds. This makes a 1.5% gel
• Once cool enough to touch added 2ul of gel green stain
• Swirled and poured into gel mould with comb
• Once solidified, covered with 1X TAE as a running buffer
• Added 1ul of purple loading dye to each of my QC strip tube samples. I had ~9ul of DNA leftover from QC and ~8ul of RNA
• Loaded my gel with the DNA first, then skipped a well and then the RNA
• Ran the gel for 60 minutes at 60 volts

Addtional Notes

• Even though qubit did not detect any gDNA in 97 or RNA in 97 there are bands on the gel